ADS-J1 Inhibits HIV-1 Entry by Interacting with gp120 and Does Not Block Fusion-Active gp41 Core Formation

We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.The essential steps of HIV-1 entry in the host cell offer several potential targets for the development of novel antiviral agents (19, 24, 33, 42). Agents that disrupt gp41-mediated membrane fusion, collectively called fusion inhibitors, were the first entry inhibitors to be approved for the treatment of HIV infection. Enfuvirtide (T20, Fuzeon) is a 36-amino-acid synthetic peptide with a sequence identical to a part of the C-terminal heptad repeat 2 (HR2) region of gp41 that binds to the N-terminal heptad repeat 1 (HR1) in an antiparallel manner, forming a coiled-coil structure during the prefusion step. Mutations in the highly conserved amino acid motif 36 to 45 in the HR1 domain confer resistance to T20 (35), providing strong evidence that HR1 is the site of interaction of T20. However, mutations in other regions of HIV-1 envelope (Env) have been also associated with T20 resistance (26, 27).Several low-molecular-weight (SMW) compounds have been identified as blockers of the initial steps of virus entry, including CCR5 coreceptor (33, 42). However, the identification of SMW compounds targeting gp41 has been elusive. A polyanionic compound, ADS-J1, was previously identified in silico as a potential candidate and shown to bind to gp41 peptides and interfere with the formation of the gp41 coiled-coil domain in an in vitro enzyme-linked immunosorbent assay (ELISA) model of HR1-HR2 interaction (16, 30, 31). Conversely, we had shown that ADS-J1 blocked the binding of HIV particles to lymphoid MT-4 cells and inhibited HIV replication at a time/site of interaction similar to those of the polyanion dextran sulfate (DS), a well-described, nonspecific inhibitor of virus entry (3). Moreover, at least four HIV-1 strains resistant to ADS-J1 were generated. The resistance to ADS-J1 was associated with gp120 based on the fact that the majority of the mutations were located in the gp120 coding sequence, mainly in the V3 loop region. Although three of the resistant strains contained mutations in gp41, one of them, HIV-1 ARA45C, did not (3). In addition, molecular modeling suggested that the gp120 V3 loop was the preferential binding site for ADS-J1 onto HIV-1, and mutations induced by the inhibitor significantly changed the stereoelectronic properties of the gp120 surface, justifying a marked drop in the affinity of ADS-J1 toward an ADS-J1-resistant HIV-1 strain (36). At that time, we considered conclusive the evidence of the mode of action of ADS-J1.More recently, Wang et al. (43) suggested that ADS-J1 could bind directly to a trimeric peptide containing the gp41 pocket region (IQN17) in a surface plasmon resonance (SPR) assay and indicated that ADS-J1 can be used as a lead compound for the design of novel HIV-1 fusion inhibitors (44). Therefore, we thought it relevant to provide further evidence of the mode of action of ADS-J1.